Structural and functional characterization of E2A:KIX interactions in leukemia

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Abstract

The E2A proteins are transcription factors critical for B-lymphopoiesis. A chromosomal translocation involving the E2A gene promotes acute lymphoblastic leukemia (ALL) through expression of the oncoprotein E2A-PBX1. Two activation domains of E2A-PBX1, AD1 and AD2, have been implicated in transcription mediated by recruitment of the transcriptional co-activator CBP/p300. A motif has been identified within AD1 that is important for recruiting CBP/p300, known as PCET. This recruitment requires an interaction between the activation domains of E2A-PBX1 and the KIX domain of CBP/p300. The KIX domain recognizes a generic ΦXXΦΦ sequence (Φ corresponds to a hydrophobic residue) found in the activation domains of numerous transcription factors. Mutation of leucine 20 in PCET has been shown to abrogate ex vivo immortalization of murine bone marrow and oncogenesis in a murine bone marrow transplantation model. A similar sequence is also found in AD2 and is implicated in E2A transcriptional activity and recruitment of CBP/p300. The structural details of these interactions remain largely unknown.
NMR spectroscopy was used to determine the solution structure of the PCET:KIX complex, and the functional consequences of the Leu20Ala mutation were structurally rationalized. Other PCET mutations informed by this structure were tested and correlations were found between in vitro binding affinities and both transcriptional activation and immortalization. The binding site of the ΦXXΦΦ-containing E2A AD2 peptide was mapped to the same site on the KIX domain used by the PCET motif. A model of this complex was generated and mutations were tested using a similar approach as was used for PCET. E2A AD2 binds the KIX domain with lower affinity than the PCET motif and is not required for immortalizing bone marrow. A mutation that increases the affinity of E2A AD2 for the KIX domain to levels approaching that seen for the PCET:KIX interaction restores transcriptional activation and immortalization, demonstrating that immortalization by E2A-PBX1 is an affinity dependent process involving the KIX domain of CBP/p300. These studies indicate that the activation domains of E2A-PBX1 serve to support the in vivo function of the oncoprotein and that the PCET:KIX complex is a potential target for novel therapeutics in E2A-PBX1+ leukemia.